Cell surface display is new technique by which a desired protein is expressed and attached onto a cell surface of microbe and is an application by using molecular biological information as the mechanism of protein secretion is elucidated. Precisely, the cell surface display uses a microbial cell surface protein derived from bacteria, yeast or the like as a surface anchoring motif to produce an exogenous protein on a cell surface. It has a broad range of application and thus, can be used to produce recombinant live vaccines, to prepare and screen a peptide/antigen library and to make whole cell absorbent, whole cell bioconversion catalyst and the like. Namely, this technique is considerably potential for an industrial use, since a variety of exogenous protein expressed onto a cell surface decides the scope of industrial application.
Cell surface carrier is the most important factor to express an exogenous protein onto a cell surface successfully. To select an effective anchoring motif expressing an exogenous protein onto a cell surface is essential for this technique.
Accordingly, the surface expression carrier makes ready for several features as follows: Above all, some secretion signal is essential to help an exogenous protein pass through a cell inner membrane and finally arrive at a cell surface. Second, a targeting signal is required to anchor an exogenous protein stably onto a cell outer surface. Third, an anchoring motif seldom affects a cell growth even if massively expressed onto a cell surface. Fourth, an exogenous protein sustains the 3-dimensional structure to be expressed stably, regardless of a protein size. However, a surface expression carrier satisfying all the terms is not found yet and is complemented only to settle some disadvantage of above cases.
In a broad sense, the surface anchoring carrier is now classified to 4 kinds, including cell outer membrane protein, lipoprotein, secretory protein, surface organelle protein such as flagella protein. In case of Gram negative bacteria, a surface protein present on a cell outer membrane such as Lam B, Pho E (Charbit et al., J. Immunol., 139: 1658-1664, 1987; Agterberg et al., Vaccine, 8: 85-91, 1990), Omp A and the like is often exploited as a surface anchoring carrier. Besides, a lipoprotein such as Tra T (Felici et al., J. Mol. Biol., 222: 301-310, 1991), peptidoglycan associated lipoprotein (PAL) (Fuchs et al., Bio/Technology, 9: 1369-1372, 1991), Lpp (Francisco et al., Proc. Natl. Acad. Sci. USA, 489: 2713-2717, 1992) and the like has been adopted and also fimbriae protein such as Fim A or Fim H adhesin of type I fimbriae (Hedegaard et al., Gene, 85: 115-124, 1989) and pili protein such as Pap A pilu subunit have been attempted to produce an exogenous protein. Furthermore, ice nucleation protein (Jung et al., Nat. Biotechnol, 16: 576-560, 1998; Jung et al., Enzyme Microb. Technol, 22(5): 348-354, 1998); Lee et al., Nat. Biotechnol., 18: 645-648, 2000), Klebsiella oxytoca pullulanase (Kornacker et al., Mol. Microl., 4: 1101-1109, 1990), Neisseria IgA protease (Klauser et al., EMBO L., 9: 1991-1999, 1990) or the like have been reported to become a surface anchoring motif. In case of Gram positive bacteria, it is known that a malaria antigen is effectively produced by using protein A derived form Staphylococcus aureus as a surface anchoring motif and that a surface coat protein from lactic acid bacteria is displayed well on a cell surface. Thus, the surface protein of Gram positive bacteria is verified to become a cell surface anchoring protein.
Previously, the present inventors have investigated a synthetic complex gene of poly-χ-glutamate, pgs BCA derived from Bacillus sp. strain, as to whether it is applicable for new surface anchoring motif. Practically, pgs BCA gene was used to develop a novel recombinant vector expressing an exogenous protein onto a microbial surface and a method for producing an exogenous protein onto a cell surface in a large scale (Korean Patent Application No. 10-2001-48373).
Furthermore, the surface expression carriers described above have been tried a lot to produce various pathogenic antigens or antigenic determinants stably through the genetic engineering technique on a highly productive bacterial surface. Especially, it is reported that an exogenous immunogen can induce more consistent and stronger immune reactions when expressed onto a non-pathogenic bacterial surface and orally administered as a live vaccine, compared with typical vaccines such as detoxified pathogenic bacteria or virus.
Since the microbial surface structure acts as an adjuvant enhancing the antigenicity of exogenous protein expressed onto a cell surface, the immune reaction is known to be induced by live bacteria within a body. It is a remarkable event to develop a recombinant live vaccine of non-pathogenic bacterium through this surface expression system.
Human papilloma virus (hereinafter, referred to as “HPV”) is presumed to world-widely infect more than 50% of all adult people. Especially, 4 types of HPV including HPV 16, 18, 31 and 45 are confirmed to cause a cervical cancer to more than 80% (Lowry D. R., Kirnbauer R., Schiller J. T., Proc. Natl. Acad. Sci., 91: 2436-2440, 1994). Papilloma virus is highly species-specific and small DNA tumor virus and belongs to family Papovaviridae which is infected to mammals such as human, cow, rabbit, sheep and so on and provokes a wart or papilloma on skin or mucosa (Pfister H., Adv. Cancer Res. 48: 113-147, 1987). Among these species, HPV are known to approximately 70 types and from more than 20 types cause tumors on skin mucosa of oral cavity or genital organs. Precisely, HPV 16 (type) and HPV 18 are reported to cause cervical cancer mostly covering women cancers.
Cervical cancer is found frequently in women, next to breast cancer world-widely. WHO (World Health Organization) has reported that cervical cancer occurring newly is over 5 hundred thousand cases every year and more than 3 hundred thousand of patients are died from cervical cancer every year in the world. Especially in the developing country, cervical cancer is a major cause of women death (Pisani P., Parkin D. M., Ferlay J., Int. J. Cancer 55: 891-903, 1993). IARC statistics showed that the most effective way to eradicate papilloma virus infection is to administer a preventive vaccine for the future, since the developing country has even more chronic patients than the advanced country.
In order to develop certain vaccine against virus, the animal culture system should be equipped properly and exploited to produce and purify virus particles in a large scale. However, HPV is hardly changed to a virus particle in vitro or in vivo, since the virion is formed only in a fully differentiated keratinocyte. Thus, there are severe problems to develop vaccines against cervical cancer for the prevention and the treatment as well as to produce virus enough for this researches. In a broad sense, 2 types of vaccine including prophylactic vaccine and therapeutic vaccine are focused as a method for producing vaccines against cervical cancer. For a purpose, the prophylactic vaccine generates a stronger neutralizing antibody by HPV L1/L2 antigen and thus prevents a host from HPV infection. Even if already infected, it makes the disease no more progressed. In the meantime, the therapeutic vaccine uses HPV E6/E7, induces specific cellular immune reactions and degenerates lesions or malignant tumor.
As searched out for last 20 years, HPV infected to human epithelium has various kinds of genotype and is associated with several benign and malignant tumors. Such an experimental data and discovery as to HPV promotes to develop HPV vaccine. HPV recombinant virus like particle is assumed more optimistic among several HPV vaccine candidates since better for an immune reaction than any other viruses except papilloma virus, through vaccine efficacy experiments in animal model and human (Koutsky L. A., Ault K. A., Wheeler C. M., Brown D. R., Barr E., Alvarez F. B., Chiacchierini L. M., Jansen K. U., N. Engi. J. of Med. 347(21): 1645-1651, 2002). Furthermore, HPV infection is confirmed recently to be the most essential cause of cancer definitely and scientists become interested in HPV studies and directly participate in this so as to accelerate the development of HPV vaccines globally. Nowadays, HPV vaccines widely known exploits HPV recombinant protein, HPV recombinant virus like particle, HPV DNA and the like to manufacture products.
In bacteria, yeast, animal cell and so on, the recombinant protein produced from some HPV partial composition through the recombinant DNA technology and a synthetic peptide in which some major epitope is synthesized chemically are tried to develop vaccines. Generally, recombinant proteins are produced through a common system such as bacteria, yeast, baculovirus, recombinant vaccinia virus and the like, by which several researches are accomplished to produce HPV recombinant proteins and to identify the antibody forming ability against HPV in serum, the induction of cellular immune reaction and the like. However, the virus system using animal and insect cells is disadvantageous and thus contaminated during cultivation and hard to be purified. Including the synthetic peptide, overall cases cost high and are commercially limited in the industry, since papilloma virus infected patients are often found in the developing countries.
Practically, HPV L1 virus like particle (hereinafter, referred to as “VLP”) has been disclosed to be produced as a live recombinant vaccinia virus through a mammary gland cell culture (Hagensee, M. E., Yaegashi, N., Gallowat, D. A., J. Virol. 67: 315-322, 1993) and VLP has been reported to generate neutralizing antibodies in a mouse model system (Schiller J. T., Lowry, D. R., Seminars in Cancer Biol. 7: 373-382, 1996). The therapeutic vaccine has been exploited by using HPV E6 and E7 protein uniquely expressed in cervical cancer (Bubenik J., Neoplasma 49: 285-289, 2002). In addition, HPV E6/E7 protein has been searched out as an immune target to treat cervical cancer and to develop a therapeutic vaccine, since it is a cancer specific antigen and associated with the cancerization of HPV infected cells. Actually, it is demonstrated that when HPV E6/E7 protein produced by the microbial system is administered to tumor cell—injected mice, the tumor formation is prevented and delayed (Gao L., Chain B., Sinclair C., J. Gen. Virol. 75: 157-164, 1994; Meneguzzi G., Cern C., Kieny M. P., Virology 181: 62-69, 1991). As shown in other cases, live virus vaccine has also problems to provoke an viral proliferation excessively and is liable to stay in a research level. Unfortunately, it takes a long time to be commercialized and also needs considerable clinical trials. In order to overcome such a disadvantage, virus vectors which are inhibited or deficient in the replication are explored but not commercialized yet (Moss B., Proc. Natl. Acad. Sci. USA 93: 11341-11348, 1996).
On the other hand, vaccine studies by using bacterial vectors are proceeded actively. It is disclosed that HPV 16 VLP produced from attenuated Salmonella typhimurium inducibly generate antigen specific antibodies in mouse mucous membrane or whole body. Besides, vaccine composed of synthetic peptides uses only essential synthetic epitope enough to induce an immune reaction for the vaccination and particularly, an epitope inducing cytotoxic T lymphocyte (CTL) against HPV 16 E6/E7 has been already elucidated (Ressing M. E., Sette A., Brandt R. M., J. Immunol. 154: 5934-5943, 1995).
In addition to such a trials, vegetables including tomatoes, potatoes and so on are utilized to produce viral antigens in plants and also a vegetable transformant itself is being attempted toward oral vaccine or edible vaccine. As a model case, hepatitis B surface antigen particle (Thavala Y. F. and C. J. Artzen. Proc. Natl. Acd. Sci. USA 92: 3358-3361) and capsid protein L1 and L2 of papilloma virus (Korean Patent Application 10-2000-0007022) are exemplified. In the plant system, however, HPV L1 protein is expressed in so small amount and so hard for the purification to limitedly engaged in commerce.
Therefore, since human papilloma virus is considered to be very often infected to people in the developing countries, in order to prevent and effectively treat tumors derived from papilloma virus onto skin mucus of oral cavity or genital organs, it is deeply required to develop a novel method for preparing human papilloma viral antigens more economically and stably.